The existence of an intracellular pathway in gingival fibroblasts for the degradation of extracellular matrix collagen has been hypothesized on the basis of in vivo ultrastructure studies showing the presence of interiorized banded collagen. This hypothesis is supported by previous in vitro microscopic studies showing that gingival fibroblasts cultured on a fibrillar collagen substrate phagocytosed the collagen into membrane-bound, lysosome-associated phagosomes in a time-dependent manner, mimicing the in vivo observations. The fate of the interiorized collagen is unknown and cannot be demonstrated using microscopic techniques. The objective of this project is to develop an in vitro system in which fibroblast-mediated intracellular collagenolysis of extracellular collagen can be directly demonstrated using biochemical techniques. The general approach will be to culture gingival fibroblast on a radiolabelled native collagen substrate, harvest the cells after significant phagocytosis has occurred, and analyze the cell layer for collagen degradation products in the form of soluble 14C peptides. Additional studies to establish the role of lysosomes in the putative intracellular collagenolysis pathway will use lysosomotropic agents to inhibit lysosomal proteinases and microtubule inhibitors to prevent phagocytosis and/or fusion of primary lysosomes with phagosomes. The proposed experimental approach is also based on biochemical studies of fibroblast-mediated intracellular degradation of newly-synthesized procollagen, on biochemical studies of hepatocytes and inflammatory cells showing lysosomal degradation of endocytosed metabolites, and on enzymologic studies showing the collagenolytic activity of lysosomal proteinase in vitro. The development of techniques to demonstrate the mechanism of fibroblast-mediated collagenolysis will permit meaningful studies directed to the regulation of tissue collagen in health and disease.